Agonist-Induced Ca[sup.2+] Signaling in HEK-293-Derived Cells Expressing a Single IP[sub.3] Receptor Isoform
In mammals, three genes encode IP[sub.3] receptors (IP[sub.3] Rs), which are involved in agonist-induced Ca[sup.2+] signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP[sub.3] R genes in HEK-293 cells, we generated three monoclonal cell lin...
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Veröffentlicht in: | Cells (Basel, Switzerland) Switzerland), 2024-04, Vol.13 (7) |
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Zusammenfassung: | In mammals, three genes encode IP[sub.3] receptors (IP[sub.3] Rs), which are involved in agonist-induced Ca[sup.2+] signaling in cells of apparently all types. Using the CRISPR/Cas9 approach for disruption of two out of three IP[sub.3] R genes in HEK-293 cells, we generated three monoclonal cell lines, IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK, with the single functional isoform, IP[sub.3] R1, IP[sub.3] R2, and IP[sub.3] R3, respectively. All engineered cells responded to ACh with Ca[sup.2+] transients in an “all-or-nothing” manner, suggesting that each IP[sub.3] R isotype was capable of mediating CICR. The sensitivity of cells to ACh strongly correlated with the affinity of IP[sub.3] binding to an IP[sub.3] R isoform they expressed. Based on a mathematical model of intracellular Ca[sup.2+] signals induced by thapsigargin, a SERCA inhibitor, we developed an approach for estimating relative Ca[sup.2+] permeability of Ca[sup.2+] store and showed that all three IP[sub.3] R isoforms contributed to Ca[sup.2+] leakage from ER. The relative Ca[sup.2+] permeabilities of Ca[sup.2+] stores in IP3R1-HEK, IP3R2-HEK, and IP3R3-HEK cells were evaluated as 1:1.75:0.45. Using the genetically encoded sensor R-CEPIA1er for monitoring Ca[sup.2+] signals in ER, engineered cells were ranged by resting levels of stored Ca[sup.2+] as IP3R3-HEK ≥ IP3R1-HEK > IP3R2-HEK. The developed cell lines could be helpful for further assaying activity, regulation, and pharmacology of individual IP[sub.3] R isoforms. |
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ISSN: | 2073-4409 2073-4409 |
DOI: | 10.3390/cells13070562 |